Heart disease is a leading cause of death in developed nations. For example, the Ministry of Health, Labour and Welfare reported heart disease as the second leading cause of death in Japan on the basis of statistical data on cause of death from 2011 to 2013.
Ischemic heart disease, which includes angina pectoris and myocardial infarction, is caused by insufficient blood flow to the myocardium due to coronary artery stenosis or coronary artery thromboembolism. In order to treat such ischemic heart disease, intervention treatment using a catheter to improve blood flow is performed. However, the myocardium cannot be regenerated by intervention treatment once necrosed. For this reason, there has been a clinical problem of an absence of an effective remedy for heart diseases including a morbid state of severe cardiac failure caused by a decrease in the number of viable cardiomyocytes after recovery from ischemia, and a morbid state of chronic progressive loss of cardiomyocytes occurring gradually that is not caused by ischemia and leads to cardiac failure.
Pluripotent stem cells have the potential of differentiating into all cells of an organism, and a typical example thereof is embryonic stem cells (ES cells). Because of such characteristics, human ES cells are expected to be applied to regenerative therapy including myocardial regenerative therapy. However, transplantation of differentiated ES cells may cause rejection, which is problematic.
In recent years, Yamanaka's group has reported the development of induced pluripotent stem cells having pluripotency and proliferation potency comparable to ES cells, in what is called iPS cells (induced pluripotent stem cells), which are prepared by inducing dedifferentiation of mouse somatic cells through expression of four factors (Oct3/4, Sox2, Klf4, and c-myc) (Non Patent Literature 1); and Yamanaka's group has subsequently reported that iPS cells can also be prepared from human differentiated cells (Non Patent Literature 2). Such human iPS cells can be prepared from cells derived from the patient to be treated, and hence are expected as a tool for the preparation of myocardial tissue without causing rejection. For this reason, there has been a demand for the establishment of myocardial regenerative therapy employing human iPS cells as early as possible.
In myocardial regenerative therapy employing human iPS cells, candidate cells serving as the source of supply include cardiomyocytes prepared by inducing human iPS cells to differentiate, and myocardial progenitor cells in the pre-differentiation stage.
When myocardial regenerative therapy is performed with cardiomyocytes prepared by inducing human iPS cells to differentiate, cardiomyocytes do not substantially proliferate and also exhibit a low take rate in the recipient's heart. For this reason, a cardiomyocyte sheet may be produced such that a sufficiently large number of cardiomyocytes for transplantation are prepared by inducing human iPS cells to differentiate, so that the sheet exhibits an increased take rate in the heart; and this cardiomyocyte sheet may be administered.
On the other hand, when myocardial regenerative therapy is performed with myocardial progenitor cells, the key is to isolate progenitor cells that specifically differentiate into the myocardium. As myocardial progenitor cells, for example, Gordon Keller et al. have reported KDR-positive PDGFRα-positive cells (Non Patent Literatures 3 and 4). However, KDR and PDGFRα, which are expressed also in early mesoderm, cannot be regarded as cell surface markers that enable identification of cell groups having the potential of specific differentiation into the myocardium. Recently, Irving L. Weissman et al. have reported that CD13 and ROR2 are cell surface markers for myocardial and vascular progenitor cells (cardiovascular progenitor cells) (Non Patent Literature 5). It is true CD13-positive ROR2-positive cells include cells that can differentiate into the myocardium; however, these cells also include cells that can differentiate into, other than the myocardium, the vascular endothelium or blood vessel walls. In addition, since expressions of CD13 and ROR2 are observed also in the primitive streak stage of early mesoderm, CD13 and ROR2 cannot be regarded as cell surface markers that enable identification of cell groups having the potential of specific differentiation into the myocardium. Furthermore, Nkx2.5, islet1, Tbx5, Tbx20, GATA4, MEF2C, and the like have been reported as markers for myocardial progenitor cells (Non Patent Literature 6). However, these markers are not cell surface markers, but transcription factors. For this reason, isolation of myocardial progenitor cells expressing such a transcription factor requires, for example, genetic modification. Thus, myocardial progenitor cells that are not modified cannot be isolated. There has been a report on a method of harvesting myocardial tissue, and directly selecting and isolating, from the tissue, pluripotent stem cells having a high potential of differentiation into cardiomyocytes (Patent Literature 1). However, since myocardial tissue is used as the source of supply, a sufficiently large number of cells for therapy may not be necessarily obtained, which raises a question of clinical application of the method. In addition, this method requires donors of myocardial tissue and hence is not very versatile. Therefore, there has been a demand for identification of a cell surface marker for myocardial progenitor cells having the potential of specific differentiation into the myocardium.